A new species of Chanohirata (Hemiptera, Cicadellidae, Deltocephalinae, Penthimiini) from southern China with its complete genome

309 A new species of Chanohirata (Hemiptera, Cicadellidae, Deltocephalinae, Penthimiini) from southern China with its complete genome Mingming Sun1* , Tielong Xu1,2*, Guy Smagghe1,3,4 , Renhuai Dai1, Xianyi Wang1,5, Jiajia Wang1,6 1 Institute of Entomology, Guizhou University, The Provincial Key Laboratory for Agricultural Pest Management Mountainous Region, Guiyang, Guizhou 550025, China 2 Guizhou Provincial Engineering Research Center of Medical Resourceful Healthcare Products, Guiyang Healthcare Vocational University, Guiyang, Guizhou, China 3 Department of Plants and Crops, Ghent University, Ghent 9000, Belgium 4 Department of Biology, Vrije Universiteit Brussels (VUB), 1050 Brussels, Belgium 5 Engineering Research Center of Medical Biotechnology, School of Biology and Engineering, Guizhou Medical University, Guiyang, Guizhou, China 6 College of Biology and Food Engineering, Chuzhou University, Chuzhou, Anhui, China Corresponding author: Renhuai Dai ([email protected]) Copyright: © Mingming Sun et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract The genus Chanohirata Hayashi & Machida, 1996 is the second most speciose genus within the tribe Penthimiini, after Penthimia Germar, 1821 and is nearly endemic to China. This study provides the description of a new species, Chanohirata pala Sun, Xu & Dai, sp. nov., discovered in Yunnan, southern China and a phylogenetic analysis including this and related taxa. The new species is diagnostically distinguished by its broad, shovel-shaped aedeagal base and a body length of 4.0–4.2 mm, with characteristic yellowish-brown coloration. A novel identification key is presented for all known Chanohirata species, based primarily on male aedeagal morphology, accompanied by a detailed geographic distribution map. Given the high morphological similarity among species within the genus, we complemented traditional taxonomy with molecular data by sequencing and assembling the complete mitochondrial genome of C. pala Sun, Xu & Dai, sp. nov. The mitogenome is 15,433 bp in length (GenBank accession number: PQ615145) and was analyzed for genome organization, base composition, relative synonymous codon usage, amino acid frequency, tRNA secondary structures, and phylogenetic relationships. Currently, Chanohirata comprises 15 species globally. Phylogenetic analyses incorporating mitochondrial protein-coding genes (PCGs), 12S rRNA and 16S rRNA, confirm that C. pala Sun, Xu & Dai, sp. nov. clusters firmly within the Deltocephalinae subfamily and is nested in the tribe Penthimiini. Notably, C. pala Sun, Xu & Dai, sp. nov. forms a sister clade with C. theae (Matsumura, 1912) and C. hamata (Wang & Zhang, 2019). This integrative approach combining detailed morphological examination with complete mitogenome sequencing advances our understanding of species boundaries and evolutionary history within Chanohirata, providing a valuable framework for future taxonomic and phylogenetic studies in Penthimiini. Key words: Checklist, mitogenome, morphology, phylogenetic, taxonomy Academic editor: J. Adilson Pinedo-Escatel Received: 30 June 2025 Accepted: 5 October 2025 Published: 12 November 2025 ZooBank: https://zoobank. org/54111796-6583-480A-93CBE38400EF0089 Citation: Sun M, Xu T, Smagghe G, Dai R, Wang X, Wang J (2025) A new species of Chanohirata (Hemiptera, Cicadellidae, Deltocephalinae, Penthimiini) from southern China with its complete genome. ZooKeys 1259: 309–333. https://doi.org/10.3897/ zookeys.1259.163605 ZooKeys 1259: 309–333 (2025) DOI: 10.3897/zookeys.1259.163605 * These authors contributed equally to the work of this paper and should be regarded as co-first authors. 310 ZooKeys 1259: 309–333 (2025), DOI: 10.3897/zookeys.1259.163605 Mingming Sun et al.: A new species of Chanohirata from China: description, key, and phylogeny Introduction Penthimiini was established by Kirschbaum (1868), it is one of the 39 tribes within the subfamily Deltocephalinae, comprising approximately 217 species across 46 genera, with a global zoogeographic distribution (Zahniser and Dietrich 2013; Wang and Zhang 2022, 2024) and it is hypothesized that the tribe Penthimiini originated in the Oriental region (Evans 1972; Cao et al. 2022), and Chanohirata most probably originated in southern China at 35.41 Ma during the late Eocene (Wang et al. 2024a), the leafhopper genus Chanohirata, established by Hayashi and Machida (1996) with Penthimia theae Matsumura, 1912 as the type species, initially included only this single species. Subsequent taxonomic and phylogenetic research has expanded the genus to 15 known species worldwide (Cheng and Li 2005; Fu and Zhang 2015; Xu and Dai 2020; Xu et al. 2020a; Wang and Zhang 2019, 2022; Wang et al. 2024a), for which we now provide an updated checklist and identification key. Mitochondrial genome (mitogenome) data have emerged as powerful tools for resolving species identification and clarifying phylogenetic relationships due to their structural and compositional stability, maternal inheritance, and limited recombination (Simon et al. 2006; Cameron 2014b; Guo et al. 2024; Wang et al. 2024b; Lu et al. 2024). Typically, insect mitogenomes range from 15 to 20 kb, comprising 22 tRNAs, 13 protein-coding genes (PCGs), two ribosomal RNA genes, and a control region (Clary and Wolstenholme 1985; Cheng et al. 2000). Within Chanohirata, morphological similarity, particularly in external features and male genitalia, poses significant challenges for species delimitation, making morphological identification alone often insufficient, for example, Viraktamath et al. (2012) misidentified Japanagallia hamata as Japanagallia neohamata due to its very similar color and form (Li et al. 2014) and species identification for taxa not reliably identified using COX1 or CYTB might be best addressed through use of multiple mitochondrial DNA fragments or other newly developed markers (Viricel and Rosel 2012). This highlights a critical research gap and the need for integrative taxonomy, combining molecular and morphological data to enhance species identification accuracy. However, only four complete mitogenomes of Penthimiini species are available in GenBank: C. hamata (Wang and Zhang 2019) (NC051985; Xu and Dai 2020), C. theae (Matsumura, 1912) (NC081953; Lv 2023), Penthimia melanocephala Motschulsky, 1863 (MT768010; Xu 2020), and Penthimia sp. (PP856705; Hassan and Xing 2025). This limited mitogenomic representation hampers comprehensive phylogenetic analyses and evolutionary studies within the genus and tribe. Expanding mitogenomic datasets is therefore essential to improve molecular taxonomy and deepen understanding of Penthimiini and Deltocephalinae evolutionary history. In this study, we describe a new species, Chanohirata pala sp. nov., based on specimens collected from Yunnan, southern China. We provide the complete mitochondrial genome sequence of this species, enhancing the molecular resources for the genus. Furthermore, we offer an updated checklist and identification key for all 16 known male Chanohirata species and investigate the phylogenetic placement of C. pala sp. nov. within Deltocephalinae using mitogenomic data. This integrative approach addresses the taxonomic challenges posed by morphological similarity and contributes to resolving the evolutionary relationships within this diverse leafhopper group. 311 ZooKeys 1259: 309–333 (2025), DOI: 10.3897/zookeys.1259.163605 Mingming Sun et al.: A new species of Chanohirata from China: description, key, and phylogeny Material and methods Taxon sampling and DNA extraction Specimens used in this study were collected using a sweep net and are deposited at the Institute of Entomology, Guizhou University, Guiyang, China (GUGC). External morphological features were examined under an Olympus SZ2-ILST stereo microscope. Adult images were captured using a Keyence VHX-6000 imaging system. Genitalia were illustrated using Adobe Illustrator CS6 and Adobe Photoshop CS6. Male genitalia were prepared by boiling in an 8–10% NaOH solution for 1.5–2.5 min, then rinsed with water and stored in glycerol. Morphological terminology and higher-level classification follow Zhang (1990), Rakitov (1997), and Dietrich (2005). Nomenclature of the new species adheres to the International Code of Zoological Nomenclature (International Commission on Zoological Nomenclature [ICZN] 1999). Total genomic DNA was extracted from the insect abdomen using the DNeasy® Blood & Tissue Kit (Qiagen, Germany). DNA purity and concentration were assessed using a Nanodrop 2000 spectrophotometer and 1% agarose gel electrophoresis. Extracted DNA was stored at -20 °C. Sequence assembly, annotation, and analysis The complete mitochondrial genome was sequenced using the Illumina HiSeq 6000 platform (Berry Genomic, Beijing, China), generating 150 bp paired-end reads with an average insert size of 350 bp. Approximately 2 GB of clean data was obtained. Raw sequence data were assembled in Geneious R9 (Kearse et al. 2012), using C. hamata (GenBank: MN922303; Xu and Dai 2020) as a reference. Genome annotation was performed using the MITOS web server (http://mitos2.bioinf.uni-leipzig.de/index.py; Donath et al. 2019). The 13 protein-coding genes (PCGs) were identified using ORF Finder in Geneious Prime with the invertebrate mitochondrial genetic code. The secondary structures of the 22 tRNA genes were predicted using tRNAscan-SE 1.21 and ARWEN 1.2 (Lowe and Eddy 1997; Laslett and Canbäck 2008). Base composition and relative synonymous codon usage (RSCU) values were calculated using MEGA X (Kumar et al. 2018), which was also used to compute amino acid usage frequency. A graphical map of the mitogenome was generated using the CGView comparison tool (https://proksee.ca; Grant et al. 2023). Strand asymmetry was calculated using the following formulas (Perna and Kocher 1995): AT skew = (A − T) / (A + T) GC skew = (G − C) / (G + C) Phylogenetic analyses To clarify the phylogenetic position of C. pala sp. nov., we analyzed its complete mitogenome along with those of 33 additional Deltocephalinae species obtained from GenBank. Two species from the subfamily Iassinae (Krisna rufimarginata and Batracomorphus lateprocessus) were used as outgroups (Table 1). 312 ZooKeys 1259: 309–333 (2025), DOI: 10.3897/zookeys.1259.163605 Mingming Sun et al.: A new species of Chanohirata from China: description, key, and phylogeny Phylogenetic reconstruction was based on sequences from 13 PCGs and two ribosomal RNA genes (12S and 16S rRNA). These sequences were extracted using Geneious Prime 2019.2.1 (Kearse et al. 2012). Multiple sequence alignment was conducted using MAFFT v7.313 within PhyloSuite v. 1.2.1. Poorly aligned regions and gaps were removed using Gblocks 0.91b (Talavera and Castresana 2007; Katoh et al. 2019; Zhang et al. 2020). Gene alignments were concatenated using MEGA X (Kumar et al. 2018). Two datasets were prepared: (i) PCG-rRNA dataset: 13 PCGs + 12S + 16S rRNA (12,105 bp), and (ii) CG-only dataset: 13 PCGs (10,875 bp). Phylogenetic trees were constructed using both Maximum Likelihood (ML) and Bayesian Inference (BI) methods based on these datasets (Huelsenbeck and Ronquist 2001; Nguyen et al. 2015). Table 1. Species, GenBank accession numbers and sources used for this study. Tribe Species GenBank accession number Acinopterini Acinopterus sp. OR187394 Acostemmini Acostemma sp.OR187395 Arrugadini Arrugada affinis NC085837 Athysanini Abrus yunshanensis NC065135 Abrus daozhenensis NC065134 Abrus expansivus NC045238 Paramacrosteles nigromaculatus NC045270 Chiasmini Nephotettix parvus NC073512 Zahniserius cylindricus NC073513 Cicadulini Cicadula sp. KX437724 Deltocephalini Alobaldia tobae KY039116 Maiestas dorsalis NC036296 Drabescini Bhatia longiradiata NC085566 Drabescoides nuchalis NC028154 Drabescus ineffectus NC050258 Roxasellana stellata NC050257 Fieberiellini Fieberiella septentrionalis NC057252 Hecalini Hecalus sp. OR187399 Iassinae (outgroup) Krisna rufimarginata NC046068 Batracomorphus lateprocessus NC045858 Mukariini Mukaria splendida NC053559 Paralimnini Paralaevicephalus gracilipenis MK450366 Yanocephalus yanonis NC036131 Penthimiini Chanohirata theae NC081953 Chanohirata hamata MN922303 Penthimia melanocephala NC051525 Chanohirata pala sp. nov. PQ615145 Scaphoideini Changbaninus pleiospicules NC060980 Phlogothamnus polymaculatus NC060774 Mimotettix multispinosus NC060773 Phlogotettix cyclops NC060772 Parascaphoidella transversa NC060771 Scaphoideus maculatus NC060770 Selenocephalini Selenocephalus sp. OR187403 313 ZooKeys 1259: 309–333 (2025), DOI: 10.3897/zookeys.1259.163605 Mingming Sun et al.: A new species of Chanohirata from China: description, key, and phylogeny Results Genome organization and nucleotide composition The complete mitochondrial genome of C. pala sp. nov. (GenBank accession no. PQ615145) is circular, with a total length of 15,433 bp (Fig. 1). It contains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and a control region (CR) rich in A+T nucleotides. Its gene arrangement and composition are consistent with those of other Cicadellidae mitogenomes (Cameron 2014a; Wang et al. 2020; Li et al. 2023; Yang et al. 2023; Lu et al. 2024; Sun et al. 2024). Among the 13 PCGs, four genes (ND1, ND4, ND4L, and ND5) are located on the N-strand, while the remaining genes (ND2, ND3, ND6, COX1, COX2, CYTB, ATP6, and ATP8) are located on the J-strand (Fig. 1), which is typical of Cicadellidae mitogenomes (Wang et al. 2017a, 2017b). The nucleotide composition of the mitogenome of C. pala sp. nov. and its components (whole genome, PCGs, rRNAs, tRNAs, and CR) is shown in Table 3: A (%): 43.2, 43.2, 43.9, 42.4, 42.5; T (%): 33.5, 32.3, 36.7, 37.5, 35.3; G (%): 9.5, 9.8, 7.5, 9.0, 10.2; and C (%): 13.8, 14.7, 11.9, 11.2, 11.6, respectively. The A+T content of the whole mitogenome is 76.7%, and the A+T content in the PCGs, rRNAs, tRNAs, and CR is 75.5%, 80.6%, 79.9%, and 77.8%, respectively (Table 3), indicating a strong A+T bias, similar to other leafhoppers (Wang et al. 2017a, 2017b, 2018). The AT-skew and GC-skew values for the genome, PCGs, rRNAs, tRNAs, and CR are as follows: AT-skew: 0.13, 0.14, 0.09, 0.06, 0.09; and GCskew: -0.18, -0.20, -0.23, -0.11, -0.06, respectively. Figure 1. Map of the mitogenome of C. pala sp. nov. 314 ZooKeys 1259: 309–333 (2025), DOI: 10.3897/zookeys.1259.163605 Mingming Sun et al.: A new species of Chanohirata from China: description, key, and phylogeny Table 2. Organization of the mitogenome of C. pala sp. nov. Locus Position Size (bp) Codon Intergenic nucleotide Strand start stop start stop anti-codon tRNA-I 1 64 64 GAT 0 F tRNA-Q 129 61 69 TTG -4 R tRNA-M 128 195 68 CAT -2 F ND2 196 1164 969 ATA TAA 0 F tRNA-W 1173 1239 67 TCA 8 F tRNA-C 1298 1231 68 GCA -9 R tRNA-Y 1366 1301 66 GTA 2 R COX1 1381 2919 1539 ATG TAA 14 F tRNA-L1 2919 2982 64 TAA -1 F COX2 2983 3663 681 ATA TAA 0 F tRNA-K 3668 3736 69 CTT 4 F tRNA-D 3736 3800 65 GTC -1 F ATP8 3801 3948 148 ATT T0 F ATP6 3950 4594 645 ATA TAA 1 F COX3 4597 5374 778 ATG T2 F tRNA-G 5375 5437 63 TCC 0 F ND3 5444 5791 348 ATA TAG 6 F tRNA-A 5789 5855 67 TGC -3 F tRNA-R 5854 5919 66 TCG -2 F tRNA-N 5917 5984 68 GTT -3 F tRNA-S1 5983 6049 67 GCT -2 F tRNA-E 6049 6116 68 TTC -1 F tRNA-F 6178 6116 67 GAA -1 R ND5 7851 6192 1660 AAC A13 R tRNA-H 7911 7851 61 GTG -1 R ND4 9232 7916 1317 TAA ATT 4 R ND4L 9495 9234 262 TAA T1 R tRNA-T 9497 9560 64 TGT 1 F tRNA-P 9625 9560 66 TGG -1 R ND6 9628 10120 493 ATT T2 F CYTB 10121 11245 1125 ATC TAA 0 F tRNA-S2 11252 11314 63 TGA 6 F ND1 12246 11308 939 TAA ATT -7 R tRNA-L2 12311 12246 66 TAG -1 R 16S-rRNA 13495 12315 1181 3 R tRNA-V 13569 13503 67 TAC 7 R 12S-rRNA 14299 13569 731 -1 R CR 14300 15433 1134 0 PCGs and codon usage The start and stop codons of the 13 PCGs, as well as the anticodons of the 22 tRNAs, are listed in Table 2. Most PCGs use typical ATN start codons (ATA, ATT, ATC, or ATG) and terminate with either TAR (TAA or TAG) or an incomplete stop codon (T). Notably, ND1 and ND4 were reported to end with ATT, and ND5 initiates with AAC and terminates with a single A, which may be sequencing artifacts or indicative of RNA editing. Among the 13 PCGs, ND5 is the longest gene (1,660 bp), and ATP8 is the shortest (148 bp), consistent with other insects (Mao et al. 2017; Wang et al. 2018; Du et al. 2019; Xu and Dai 2021). 315 ZooKeys 1259: 309–333 (2025), DOI: 10.3897/zookeys.1259.163605 Mingming Sun et al.: A new species of Chanohirata from China: description, key, and phylogeny Figs 2, 3 summarize the relative synonymous codon usage (RSCU) and amino acid frequency. The most frequently used codon is CGA (Arg), followed by UUA (Leu), whereas the least used is UCG (Ser1). The most frequently encoded amino acids are Ser, Leu, Lys, Asn, and Ile. The prevalent use of codons composed entirely of A or T reflects the high A+T content of the genome. tRNAs, rRNAs, and CR All 22 tRNAs were predicted using tRNAscan-SE 1.21, ARWEN 1.2, and MITOS2. Most tRNAs exhibit the typical cloverleaf secondary structure, except for trnS1, which lacks the dihydrouridine (DHU) arm and instead forms a simple loop, with is a common feature in many insects (Sheffield et al. 2008; Cao and Du Figure 2. RSCU in the mitogenome of C. pala sp. nov.; the stop codon is not included. Figure 3. Overall amino acid usage frequency in the mitogenome of C. pala sp. nov. 316 ZooKeys 1259: 309–333 (2025), DOI: 10.3897/zookeys.1259.163605 Mingming Sun et al.: A new species of Chanohirata from China: description, key, and phylogeny 2014; Wang et al. 2014; Zhang et al. 2014; Wang et al. 2017a, b). Additionally, trnG lacks the TΨC arm (Fig. 4). The lengths of the tRNAs range from 61 bp (tRNA-His) to 69 bp (tRNA-Gln) (Table 2). Several base mismatches were observed in tRNA secondary structures (Table 4), including: A-A mismatches (e.g., in trnE), U-U mismatches (e.g., in trnQ, trnL1, trnL2, trnW, and trnR), and G-U mismatches, which were most frequent, observed in 13 tRNAs (trnA, trnD, trnQ, trnG, trnH, trnL1, trnK, trnF, trnP, trnS1, trnW, trnY, trnV). These findings suggest that further research on tRNA secondary structures is warranted. As in other insect mitogenomes, C. pala sp. nov. contains two rRNA genes: rrnL (1,181 bp) and rrnS (731 bp) (Table 2). Their locations are conserved: rrnL lies between trnL2 and trnV, and rrnS lies between trnV and the control region (CR) (Table 2, Fig. 1). The CR (also called the A+T-rich region) is the longest non-coding segment and varies greatly among species in both length and sequence composition, which is a key factor in mitogenome diversity among leafhoppers (Wang et al. 2020). In C. pala sp. nov., the CR is 1,134 bp in length, located between rrnS and trnI, with an A+T content of 77.8% (Tables 2, 3). Phylogenetic analysis The phylogenetic relationships of Deltocephalinae were inferred using 34 species based on two datasets: (i) 13 protein-coding genes (PCGs) and two ribosomal RNA genes (12S and 16S rRNA), comprising 12,105 nucleotides (PCG+rRNA); and (ii) PCGs only, comprising 10,875 nucleotides. Four phylogenetic trees were generated using Bayesian Inference (BI) and Maximum Likelihood (ML) methods (BI-PCGs, BI-PCG+rRNA, ML-PCGs, ML-PCG+rRNA), all of which yielded similar topologies. Table 4. Total numbers of different types of base mismatches in tRNAs. Species G-U U-U A-A C. pala sp. nov. trnA (1) TrnD (2) TrnQ (1) TrnG (2) TrnH (2) trnQ (1) TrnL1 (1) TrnL1 (1) TrnK (2) TrnL2 (2) trnE (1) TrnF (2) trnW (1) TrnP (1) trnR (2) TrnS1 (1) TrnW (1) TrnY (1) TrnV (3) Table 3. Nucleotide composition of the C. pala sp. nov. mitogenome. Length (bp) A% C% T% G% A+T% AT-skew% GC-skew% Genome 15433 43.2 13.8 33.5 9.5 76.7 0.13 -0.18 PCGs 10904 43.2 14.7 32.3 9.8 75.5 0.14 -0.20 rRNA 1912 43.9 11.9 36.7 7.5 80.6 0.09 -0.23 tRNA 1449 42.4 11.2 37.5 9.0 79.9 0.06 -0.11 CR 1134 42.5 11.6 35.3 10.2 77.8 0.09 -0.06 317 ZooKeys 1259: 309–333 (2025), DOI: 10.3897/zookeys.1259.163605 Mingming Sun et al.: A new species of Chanohirata from China: description, key, and phylogeny Previous studies have examined the phylogenetic relationships within Deltocephalinae using either morphological or molecular data (Zahniser and Dietrich 2008, 2010, 2013; Du et al. 2017a, 2017b; Hassan et al. 2023). In our study, mitogenomic data produced well-resolved trees supporting several established relationships. The results indicate that Penthimiini occupies a basal position in the phylogenetic tree (Figs 5, 6; Suppl. material 1: figs S1, S2), which is consistent with previous findings (Zahniser and Dietrich 2013; Wu et al. 2021). C. pala sp. nov. clusters with other Deltocephalinae species and specifically with C. theae and C. hamata within Penthimiini. Within Deltocephalinae, Scaphoideini forms a distinct clade with Drabescini, with both lineages recovered as sister groups (Figs 5, 6; Suppl. material 1: figs S1, S2), corroborating previous findings by Zahniser and Dietrich (2013), Xu et al. (2020b), and Lu et al. (2023). Stable sister-group relationships were also observed between Deltocephalini and Paralimnini, and between Selenocephalini and Cicadulini. However, the phylogenetic placement of Athysanini remained unresolved and unstable across datasets, echoing earlier studies (Wu et al. 2021; Hassan et al. 2023). In addition, the monophyly of Chiasmini, Deltocephalini, Drabescini, Paralimnini, Penthimiini, and Scaphoideini was strongly supported in all analyses (Figs 5, 6; Suppl. material 1: figs S1, S2). Figure 4. Predicted secondary structures of 22 tRNAs. 324 ZooKeys 1259: 309–333 (2025), DOI: 10.3897/zookeys.1259.163605 Mingming Sun et al.: A new species of Chanohirata from China: description, key, and phylogeny Etymology. The specific epithet pala is derived from Latin, meaning shovel, referring to the broad, shovel-shaped base of the aedeagus. Remarks. This species is similar to C. dactyla but can be distinguished by the shape of the aedeagus. Discussion The primary goal of this study was to describe a new species, C. pala sp. nov., collected from Yunnan, China, investigate the structural characteristics of its mitochondrial genome and phylogenetic placement within the tribe Deltocephalinae. While previous studies have explored the phylogeny of Deltocephalinae using morphological and molecular data (Zahniser and Dietrich 2013; Wang and Xing 2019; Luo et al. 2021; Wu et al. 2021; Yang and Dai 2021; Cao et al. 2022; Lu et al. 2023; Hassan et al. 2023; Wang et al. 2024a), but focused research on the tribe Penthimiini remains limited. To date, only one phylogenetic reconstruction of the Figure 9. Distribution map of Chanohirata species from China. 325 ZooKeys 1259: 309–333 (2025), DOI: 10.3897/zookeys.1259.163605 Mingming Sun et al.: A new species of Chanohirata from China: description, key, and phylogeny Figure 10. Male aedeagus of Chanohirata. A. C. citrana lateral view (Cheng & Li, 2005); B. C. spinata lateral view (Cheng & Li, 2005); C. C. minima dorsal view (Wang & Zhang, 2022) ; D. C. yunnana ventral view (Wang & Zhang, 2022); E. C. plania dorsal view (Wang & Zhang, 2022); F. C. dactyla dorsal view (Fu & Zhang, 2015); G. C. eurya dorsal view (Fu & Zhang, 2015); H. C. theae ventral view (Fu & Zhang, 2015); I. C. lini dorsal view (Cheng & Li, 2005); J. C. lageniformia posterior view (Wang & Zhang, 2019); K. C. hamata posterior view (Wang & Zhang, 2019); L. C. bipennata dorsal view (Xu et al. 2020a); M. C. cornicula ventral view (Wang & Zhang, 2024a); N. C. serrata ventral view (Wang & Zhang, 2024a); O. C. elongata ventral view (Wang & Zhang, 2024a); P. C. pala. sp. nov. dorsal view. 326 ZooKeys 1259: 309–333 (2025), DOI: 10.3897/zookeys.1259.163605 Mingming Sun et al.: A new species of Chanohirata from China: description, key, and phylogeny genus Chanohirata has been conducted (Wang et al. 2024a), and only four complete mitochondrial genomes from Penthimiini are available in GenBank. This study contributes a new complete mitochondrial genome for C. pala sp. nov., significantly expanding the mitogenomic resources for the tribe. Our phylogenetic analysis based on mitochondrial protein-coding and rRNA genes successfully recovered several tribes within Deltocephalinae as monophyletic, including Chiasmini, Deltocephalini, Drabescini, Paralimnini, Penthimiini, and Scaphoideini, while Athysanini was not supported as monophyletic, these results are consistent with earlier findings by Hassan et al. (2023). Phylogenetic analyses based on mitochondrial genes confirmed that C. pala sp. nov. clusters with other Deltocephalinae species and belongs to the genus Chanohirata, forming a clade with C. theae and C. hamata; therefore, we speculate that the origin of C. pala sp. nov. was in southern China during the late Eocene (Evans 1972; Cao et al. 2022; Wang et al. 2024a). However, the monophyly and interrelationships of several other tribes, such as Athysanini, Acinopterini, Acostemmini, Arrugadini, Fieberiellini, Hecalini, Mukariini, and Selenocephalini, remain unresolved due to a lack of comprehensive mitogenomic data. Nonetheless, the major research advances of this study include the formal description of a new species with detailed morphological and molecular data, the first integrated mitogenomic analysis of C. pala sp. nov., and support for the monophyly of several key tribes within Deltocephalinae based on expanded genomic data. We believe that innovative aspects include the use of complete mitogenomic sequencing to overcome the limitations of morphology-based identification in a morphologically conserved genus. Future directions should prioritize broader taxon sampling and the generation of more mitogenomic datasets for Deltocephalinae. Increasing the number of complete mitochondrial genomes will enhance phylogenetic resolution and enable a more comprehensive understanding of evolutionary patterns within this diverse subfamily. Supplementing existing molecular data is thus critical to clarifying the deep relationships among leafhopper lineages. In addition, we examined several mitochondrial features of C. pala sp. nov., including genome organization, base composition, relative synonymous codon usage, amino acid usage, and tRNA secondary structures. These characteristics were largely consistent with patterns observed in previous leafhopper mitogenome studies (Wu et al. 2016; Yu et al. 2017; Wang et al. 2017a, 2017b, 2019, Wang and Xing 2019; Mao et al. 2017; Du et al. 2017a, 2019; Lu et al. 2024; Sun et al. 2024). Currently, the genus Chanohirata comprises 16 known species, which can be distinguished based on differences in aedeagal morphology (Fig. 10). Most species are distributed in southwestern China, with a few extending into adjacent regions (Fig. 9). This study underscores the importance of combining molecular and morphological approaches in taxonomic research. The integrative framework presented here offers new insights into the systematics and evolutionary history of Chanohirata, setting the foundation for further phylogenetic and biogeographic studies within the Penthimiini and broader Deltocephalinae. Conclusions In this study, we described and conducted a phylogenetic analysis of a new species, C. pala sp. nov., from Yunnan Province, southern China. The results highlight the significance of integrating molecular and morphological data in 327 ZooKeys 1259: 309–333 (2025), DOI: 10.3897/zookeys.1259.163605 Mingming Sun et al.: A new species of Chanohirata from China: description, key, and phylogeny taxonomic studies. Phylogenetic analyses based on mitochondrial genes confirmed that C. pala sp. nov. clusters with other Deltocephalinae species and belongs to the tribe Penthimiini, forming a clade with C. theae and C. hamata. We also sequenced and assembled the complete mitogenome of C. pala sp. nov., which is 15,433 bp in length and available under GenBank accession number PQ615145. Mitochondrial features such as genome organization, base composition, codon usage, amino acid frequency, and tRNA secondary structures were characterized and found to be consistent with those of related species. However, the monophyly and interrelationships of some Deltocephalinae tribes remain unresolved due to insufficient mitogenome data. Therefore, expanding molecular datasets is critical for future phylogenetic studies and for refining the taxonomy and evolutionary understanding of Deltocephalinae. Acknowledgements We sincerely thank Meisu Guo, Xiaozhen Lu, Siying You, and Hongyun Chen for collecting the specimens used in this study. We are also grateful to Jiapeng Yang and Yunfei Wu for their guidance on illustration techniques. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Use of AI No use of AI was reported. Funding This research was supported by the National Natural Science Foundation of China (No. 32160119); the National Natural Science Foundation of China (No. 32000329); the Program of Excellent Innovation Talents, Guizhou Province, China (No. 20206003-2); and the Opening Foundation of Shanxi University of Technology (No. SLGPT2019KF03-01). The following grant information was disclosed by the authors: National Natural Science Foundation of China: No. 32160119. Program of Excellent Innovation Talents, Guizhou Province, Chin: Grant number 20206003-2. Author contributions Mingming Sun conceived and designed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft. Tielong Xu performed the experiments, analyzed the data, authored or reviewed drafts of the article, and approved the final draft. Guy Smagghe authored or reviewed drafts of the article, and approved the final draft. Renhuai Dai conceived and designed the experiments, performed the experiments, authored or reviewed drafts of the article, and approved the final draft. Xianyi Wang conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft. Jiajia Wang conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft. 328 ZooKeys 1259: 309–333 (2025), DOI: 10.3897/zookeys.1259.163605 Mingming Sun et al.: A new species of Chanohirata from China: description, key, and phylogeny Author ORCIDs Mingming Sun https://orcid.org/0009-0000-3702-109X Guy Smagghe https://orcid.org/0000-0001-8334-3313 Renhuai Dai https://orcid.org/0009-0003-2118-9316 Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. The following information was supplied regarding the deposition of DNA sequences: Chanohirata pala sp. nov.: PQ615145. References Cameron SL (2014a) Insect mitochondrial genomics: Implications for evolution and phylogeny. 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The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1259.163605.suppl1